The proposed studies are part of our long-range effort to study the fundamental mechanisms of transcription and its regulation using bacterial model systems. Proper control of gene regulation is critical for organismal development, cellular response to environmental signals, and the prevention of disease states. The first step in gene expression, transcription, is carried out by multi-subunit RNA polymerases (RNAPs) that are conserved in sequence, structure and function from bacteria to humans. Thus, a detailed mechanistic understanding of transcription in bacteria facilitates an understanding of transcription in all organisms. The proposed studies will apply a comprehensive approach combining genetics, biochemistry, structural biology, genomic, and high- throughput sequencing based methods to (1) understand the diversity of regulatory mechanisms that link changes to cellular state to changes in the activity of RNAP and (2) obtain a quantitative understanding of transcription that defines the mechanistic basis for each step of transcription and confers the ability to predict transcriptional output from the DNA sequence content of a gene. We tackle these areas by developing new methodologies to understand the interface between DNA sequence and RNAP function, and to reexamine long-held notions of RNAP function for unexpected paradigms or unknown biology.